The present invention relates to a system that analyzes biological substances and the like by a magnetic signal by using magnetic nano particles and a magnetic sensor, for example, an immunoassay system used for inspection of, for example, immunoglobulin, hormone, a tumor marker, infectious diseases or the like, a blood culturing device and a bacterial culturing device used for inspection of pathogenic bacterium of an infectious disease, food poisoning and the like.
Inspection of various biological substances (hormone, antibody, antigen, tumor markers and the like), pathogenic bacterium, viruses, cancer cells, DNA, environmentally hazardous materials and the like is possible by using specific binding reaction such as antigen-antibody reaction. In recent years, there has been a growing demand for quick and highly sensitive detection of these inspection targets, and the immunoassay systems for this purpose have been energetically developed. As a general method of immunoassay, there is an optical method in which an antibody for detection which selectively adheres to the antigen to be a detection target is marked with an optical marker such as a luminescence enzyme or the like, the binding reaction of antigen and the antibody is detected by detecting an optical signal from the optical marker, and the kind and quantity of the antigen are detected. However, in the optical method, the detection sensitivity is not sufficient, and the process of washing the unbound optical marker (washing process) is required.
In recent years, a magnetic method for detecting an antigen-antibody reaction by using magnetic nano particles and a magnetic sensor has been proposed. In the magnetic method, an antibody magnetically marked with magnetic nano particles (hereinafter, called a magnetic marker) is bound to the substance of the detection target, and the magnetic signal generated from the bound magnetic marker is detected by using a magnetic sensor. By using a Superconducting quantum interference device (SQUID) which is a highly sensitive magnetic sensor, detection sensitivity higher than that of the optical method is obtained.
There are proposed various methods for detecting biomaterials by magnetic signals with magnetic nano particles as labels.
In the method disclosed in JP-61-235774, a substance to be measured is fixed to a container and thereafter, is reacted with a magnetic marker, and after the unbound magnetic marker is washed, a residual magnetic signal from the magnetic marker which is bound to the substance to be measured fixed to the container is measured with a SQUID.
In the method disclosed in JP-2-15551, by mixing the solution containing a substance to be measured and magnetic markers in a solution, the substance to be measured and the magnetic markers are bound to form an aggregate, and after in the state where unbound magnetic markers exist, an external magnetic field is applied to align the directions of the magnetic markers, the magnetic signal after the magnetic field is shut off is measured. The directions of the magnetic moments of the magnetic markers in suspension in the solution become random due to Brownian motion, and therefore, even when the directions of the magnetic moments are aligned by the magnetic field, if the magnetic field is shut off, the magnetic signal gradually decays. When the magnetic markers bind with the target substance and aggregate, the volume as a rotating body increases, and therefore, decaying time of the magnetic signal is delayed, but the magnetic signal of the unbound magnetic marker decays early. Since the relaxation time constant of the magnetic signals differ, the magnetic signal of the bound marker can be measured without removing the unbound marker. Similar methods are also reported in Y. R. Chemla, et al.: Proc. National Acad. Sciences of U.S.A. 97, 14268 (2000), A. Haller, et al.: IEEE Trans. Appl. Supercond. 11, 1371 (2001), H. L. Grossman, et al.: Proc. National Acad. Sciences of U.S.A. 101, 129 (2004) and JP-A-10-513551.
In the methods of JP-A-2005-257425 and K. Enpuku, et al.: IEEE Trans. Appl. Supercond. 13, 371 (2003), the substance to be measured is fixed to the container, and thereafter, caused to react with a magnetic marker, and a signal from the magnetic marker bound to the substance to be measured which is fixed to the container in the state in which an unbound magnetic marker exists is measured with a SQUID magnetic sensor. The directions of the magnetic moments of the unbound magnetic markers become random due to Brownian motion, and therefore, the magnetic signals decay. Therefore, the signal from the bound magnetic marker can be measured without washing the unbound marker. A similar method is also reported in R. Kotitz, et al.: IEEE Trans. Appl. Supercond. 7, 3678 (1997).
In JP-A-2001-33455 and JP-A-2001-133458, methods using susceptibility measurement are reported. In JP-A-2001-33455, a DC magnetic field which magnetizes the magnetic marker is applied from the direction orthogonal to the magnetic flux detecting direction of a SQUID magnetic sensor, and the change in the magnetic field caused by the magnetic markers which move in the magnetic flux detection region of the SQUID magnetic sensor is measured. In JP-A-2001-133458, an AC magnetic field is applied to the magnetic marker, and antigen-antibody reaction is detected by detecting the signal by using a SQUID magnetic sensor.
As above, magnetic signals from the bound magnetic markers are measured by the various methods, but the measured magnetic signals include external ambient magnetic signals, magnetic signals from magnetic impurities included in the containers, and magnetic signals from the magnetic markers nonspecifically bound to the containers, as noise signals. In order to make highly accurate measurement, it is necessary to reduce these magnetic signals.
As described above, the measured magnetic signals include not only signals from the bound magnetic marker and unbound magnetic marker, but also a noise signal of the sensor itself, external ambient magnetic signals, magnetic signals from the magnetic impurities included in the container, and magnetic signals from the magnetic markers nonspecifically bound to the container. In order to make measurement with high accuracy, these magnetic signals need to be reduced.
In the manufacturing process of the container, it is extremely difficult to suppress inclusion of magnetic impurities completely even if the containers are produced with close attention paid to the raw material, production process and the like. Even if magnetic impurities are not included in the container itself, dust and the like which are magnetized are likely to adhere to the container bottom surface and the like. In the optical method, such contamination of the container does not become a problem. The substance used for an optical marker such as phosphors does not exist in an ordinary manufacturing process and use environment, and is not included in or does not adhere to the container. However, in an ordinary environment, a number of substances having magnetism exist. Therefore, impurities and contamination of the container are new problems peculiar to magnetic detection.
Meanwhile, nonspecific binding of markers to the containers also becomes a problem in optical methods. The container surface is usually coated with a blocking agent such as a BSA for preventing nonspecific binding, but it is difficult to prevent nonspecific binding of a marker completely. Especially in the inspection system of the method in which the measuring container is reused by washing, for example, of a flow cell method, washing is performed a plurality of times in order to remove the marker adhering to the measuring container, but it is difficult to remove it completely with favorable reproducibility, and this becomes the factor that restricts the lower limit concentration of detection.